EXAM3 : data interpretation

Question 1

what are the steps in STR genotyping / interpretation

Answer 1

• peak detection/interpretation thresholds
• peak sizing
• DNA size (allele call)
• DNA fragment analysis

Question 2

what does internal size standard do?

Answer 2

it runs along with the sample and it shows the size vs time of DNA fragments

Question 3

___ is the bin defined around each allele

Answer 3

+/- 0.5

Question 4

what is allele call?

Answer 4

determined by comparison of
peak size (bp) to allelic ladder
allele peak sizes run under the
same electrophoretic conditions

Question 5

what is the detection threshold ?

Answer 5

50-200 RFU

Question 6

why do we use thresholds?

Answer 6

to separate signal from noise (is the peak real or not)

Question 7

analytical RFU ____

Answer 7

50

Question 8

Interpretation RFU

Answer 8

150 RFU

Question 9

what is signal peak height measured in ? what is it related to?

Answer 9

Relative Fluorescence Units (RFU)
- related to the amount of DNA present in the sample loaded onto the analysis instrument

Question 10

what 3 issues impact mixture interpretation?

Answer 10

1.) peak detection threshold
2.) peak height ratio
3.) stutter percentage

Question 11

biological artifact vs technology artifact issues

Answer 11

B: stutter, “N” peak, Off ladder, Tri-Alleles
T: Pull up, Dye blobs, air bubbles, urea crystals/particles, and voltage peaks

Question 12

what happens during allelic drop-outs?

Answer 12

degradation and inhibition of the primer binding site mutation

Question 13

what are the data interpretation issues

Answer 13

1. artifact peaks vs allele peaks
2. allelic drop outs
3. mixtures
4. mutations

Question 14

what are stutter products ?

Answer 14

peaks that show up primarily one repeat less than the true allele

Question 15

how does stutter happen?

Answer 15

a result of strand slippage during DNA synthesis

Question 16

which repeat regions generate more stutter?

Answer 16

longer

Question 17

where is stutter less pronounced ?

Answer 17

larger repeat unit sizes

Question 18

true or false: each successive stutter product is less intense

Answer 18

true

Question 19

how much of the true allele in tetranucleotides repeats STR loci

Answer 19

5-15%

Question 20

(N-4) stutter product is …

Answer 20

Deletion - caused by slippage on the copied BOTTOM strand

Question 20

(N+4) stutter product is …

Answer 20

Insertion - caused by slippage of the copying TOP strand

Question 21

In strand slippage, the lower incorporation rate allows what?

Answer 21

more opportunity for the DNA strands to breathe apart during PCR (same mechanism as mutation)

Question 22

Taq Polymerase - stutter peaks

Answer 22

50-60 nt/second

Question 23

Stoffel fragment - stutter peaks

Answer 23

5-10 nt/second

Question 24

Is a "stutter" the same size as an allele ?

Answer 24

YES !

Question 25

what is the average stutter in D3S1358/ vWA?

Answer 25

7%

Question 26

what is the average stutter for TPOX?

Answer 26

3%

Question 27

percent stutter tends to increase with __

Answer 27

allele length

Question 28

the proportion of stutter to main allele is ...

Answer 28

generall reproducible within a locus and even for a given allele length

Question 29

alleles with longer core repeat unit regions generally have _____ proportion of stutter

Answer 29

increase

Question 30

alleles with interrupted core repeat regions tend to have ___ proportion of stutter

Answer 30

less

Question 31

what are the strategies for stutter reduction?

Answer 31

- cosolvents: osmolytes - cycling parameters - mutants of taq polymerase

Question 32

In non-template addition what does taq polymerase do?

Answer 32

it will often add an extra nucleotide to the end of a PCR product , often "A" (adenlyation)

Question 33

why is non-template addition dependent on the 5'-end of the reverse primer?

Answer 33

it is dependent on this end so that a "G" can be put at the end of a primer to promote the non-template addition

Question 34

how can the non-template addition be enhanced?

Answer 34

with extension soak at the end of the PCR cycle ( 15-45 min @60 or 72 degrees to give polymerase more time)

Question 35

excess amounts of DNA template in the PCR reaction can result in...

Answer 35

incomplete adenylation (not enough polymerase to go around)

Question 36

why do we NOT want a mixture of (+/- A peaks)

Answer 36

to avoid split peaks

Question 37

What observations indicate that a peak in an electropherogram is a spike as opposed to DNA?

Answer 37

sharp peak, that shows up in all of the panels (spike), where the allele is more curved

Question 38

Is it better to prevent or promote non-template addition? Explain your reasons?

Answer 38

promote, to get it to A+ , we want it to be A+!!

Question 39

What steps are typically taken to confirm the presence of an off- ladder allele?

Answer 39

does not match up with allelic ladder, meaning it doesn't show up in the ladder ** if it is off then you would run it again, and again - sometimes there is a temperature difference in your sample vs ladder which would mess up with alignment - if you keep seeing it then it is off ladder